 1998 Oxford University Press
2694–2701 Nucleic Acids Research, 1998, Vol. 26, No. 11
Thermodynamics of internal C·T mismatches in DNA
Hatim T. Allawi and John SantaLucia, Jr*
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received February 6, 1998; Revised and Accepted April 6, 1998
ABSTRACT
Thermodynamics of 23 oligonucleotides with internal
single C·T mismatches were obtained by measuring
UV absorbance as a function of temperature. Results
from these 23 duplexes were combined with three
measurements from the literature to derive nearestneighbor thermodynamic parameters for seven linearly
independent trimer sequences with internal C·T mismatches. The data show that the nearest-neighbor model
is adequate for predicting thermodynamics of oligonucleotides with internal C·T with average deviations for
∆G37, ∆H, ∆S and Tm of 6.4%, 9.9%, 10.6%, and 1.9C
respectively. C·T mismatches destabilize the duplex in
all sequence contexts. The thermodynamic contribution
of C·T mismatches to duplex stability varies weakly
depending on the orientation of the mismatch and its
context and ranges from +1.02 kcal/mol for GCG/CTC
and CCG/GTC to +1.95 kcal/mol for TCC/ATG.
derived nearest-neighbor thermodynamic parameters for internal
G·T and G·A mismatches and showed that, when combined with
the thermodynamics of Watson–Crick pairs, accurate predictions
of thermodynamics of duplexes with G·T and G·A mismatches
can be determined with average standard deviations for ∆G37,
∆H, ∆S and Tm of 5%, 8%, 8%, and 1.5C respectively
(17,21). To add to our mismatch parameter database and to test
whether the nearest-neighbor model is applicable to unstable
mismatches, such as C·T mismatches (22–24), we obtained
thermodynamic measurements of 28 DNA duplexes containing
internal C·T mismatches and combined them with three literature
values (24,25) to derive nearest-neighbor parameters for internal
C·T mismatches in DNA. The availability of internal C·T
mismatch nearest-neighbor parameters along with Watson–Crick
nearest-neighbors allows reliable prediction of duplex stability
from sequence.
MATERIALS AND METHODS
Absorbance versus temperature melting curves
INTRODUCTION
DNA mismatches occur as a result of errors during replication (1),
due to heteroduplex formation during homologous recombination
(2) and mutagenic chemicals and ionizing radiation or spontaneous
deamination (3). Mismatches also occur in the secondary
structures of single-stranded DNA viruses (4–6). In addition to
stable canonical Watson–Crick base pairs (G·C and A·T) there are
eight possible mispairs of varying stability and structure, namely
A·A, A·C, C·C, C·T, G·G, G·A, G·T and T·T. In order to
understand the origins of various mismatch occurrences and to
help in our interpretation of mismatch recognition and repair
mechanisms, thermodynamics and structures of these mismatches
need to be determined.
Several molecular biological techniques require accurate
predictions of matched versus mismatched hybridization
thermodynamics, such as PCR (7), sequencing by hybridization
(8), gene diagnostics (9) and antisense oligonucleotide probes
(9–11). In addition, recent developments of oligonucleotide chip
arrays as means for biochemical assays and DNA sequencing
requires accurate knowledge of hybridization thermodynamics
and population ratios at matched and mismatched target sites
(8,12,13).
We and others showed that a nearest-neighbor model is
sufficient to accurately predict the stability and thermodynamics
of DNAs with Watson–Crick pairs (14–20). Thereafter, we
Oligonucleotides were synthesized on solid supports using standard
phosphoramidite techniques (26) and deblocked and purified as
described previously (17). Absorbance versus temperature profiles
were determined using an AVIV 14DS UV-vis spectrophotometer
with a heating rate of 0.8C/min as described previously (16).
Oligonucleotides were dissolved in 1.0 M NaCl, 20 mM sodium
cacodylate and 0.5 mM Na2EDTA, adjusted to pH 7.0 or 5.0 with
1.0 M HCl. Prior to the beginning of each melt, the samples were
annealed and degassed by raising the temperature to 85C for 5 min
and then slowly cooling the samples to –1.5C. While at high
temperature, oligonucleotide absorbances at 260 nm were
recorded and used to calculate single-strand total concentrations
(CT) using extinction coefficients calculated for dinucleoside
monophosphates and nucleosides (27). Absorbance melting
curves for each duplex were measured at 260 and 280 nm from
0 to 85 or 90C at 8–10 different concentrations.
Data analysis
Thermodynamic parameters for duplex formation were obtained
from UV melting curves using the program MELTWIN v2.1 (28)
assuming a two-state transition (i.e. duplex and random coil) by
two methods: (i) averages of ∆H and ∆S from fits of 8–10
melting curves at different concentrations as described (29);
(ii) plots of reciprocal melting temperature (Tm–1) versus lnCT
according to the equation (30)
*To whom correspondence should be addressed. Tel: +1 313 577 0101; Fax: +1 313 577 8822; Email: jsl@chem.wayne.edu
2695
Nucleic Acids
Acids Research,
Research,1994,
1998,Vol.
Vol.22,
26,No.
No.111
Nucleic
Tm –1 = R/∆H ln(CT/N) + ∆S/∆H
1
For self-complementary sequences, N = 1 and for non-selfcomplementary sequences, N = 4. For the two-state model to apply,
agreement of the parameters obtained using the two different
methods is a necessary, but not sufficient, condition (17,31).
Sequences design and rationale
Sequences were designed to have a melting temperature between
30 and 55C and to minimize the potential of forming alternative
competing secondary structures (i.e. hairpins or ‘slipped’ duplexes),
which maximizes the likelihood of observing two-state transitions.
Throughout this paper nearest-neighbors are represented in an
antiparallel fashion with a slash separating the two stands and an
underline indicating the position of C·T mismatches. For
example, the sequence AC/TT means 5′-AC-3′ paired with
3′-TT-5′. In this study, the eight different C·T mismatch-containing
dimers are evenly represented and occur with the following
frequencies: AC/TT = 6, AT/TC = 9, CC/GT = 9, CT/GC = 8,
GC/CT = 10, GT/CC = 9, TC/AT = 9, TT/AC = 8. In addition, all
16 possible Watson–Crick surrounding contexts are represented
at least once in the data set.
Determination of C·T mismatch contribution to duplex
stability
Van’t Hoff analysis of melting curves provides total ∆G37, ∆H
and ∆S for the duplex to random coil transition. Applying the
nearest-neighbor model to each duplex allows determination of
the internal C·T mismatch contribution to duplex stability. For
example, the internal C·T mismatch contribution to the duplex
GGACCGACG·CGTCTGTCC [which has a ∆G37(expt) of
–6.58 kcal/mol] is the sum of initiation and nearest-neighbor
propagation terms
5-GGACCGACG-3
3-CCTGTCTGC-5 GG GA AC CC CT GA AC CG
Init. CC CT TG GT GC CT TG GC 2
Note that for self-complementary sequences a symmetry penalty
is also included in the calculation (32). The C·T mismatch
contribution, ∆G37(mismatch), is calculated by rearranging
equation 2 to give
5-GGACCGACG-3
CC CT
GT GC 3-CCTGTCTGC-5 –
GG GA AC GA AC CG
Init.– CC – CT – TG – CT – TG – GC
3
Substitution of nearest-neighbor parameters for Watson–Crick
and initiation terms, which have been previously determined
(17), into equation 3 gives
CC CT
GT GC –6.58 – (1.96) – (–1.84) – (–1.30) – (–1.44) – (–1.30) – (–1.44) – (–2.17)
∆G37(mismatch) = ∆G37(CC/GT + CT/GC) = + 0.95 kcal/mol 4
Therefore, the C·T mismatch in the context CCG/GTC destabilizes
the free energy of duplex formation by 0.95 kcal/mol. Similar
2695
calculations are also performed for ∆H and ∆S to determine
∆H(mismatch) and ∆S(mismatch).
Determination of thermodynamics of linearly independent
sequences with C·T mismatches
The contribution of a mismatch to DNA duplex stability depends on
the location of the mismatch, its nearest neighbors and its orientation
(17). A mismatch located in the middle of the duplex is less stable
than a mismatch located at the termini (17). For C·T mismatches,
imposing a restriction on the location of the mismatch, such as
forming all internal C·T mismatches, reduces the number of
independent parameters that can be derived from the data set from
eight to seven (17,33). Instead of the usual dimer sequences format
for nearest-neighbor parameters, internal mismatches should be
represented in terms of trimer sequences with the mismatch being
in the middle position. There are 16 unique possibilities for such
trimer duplexes with internal C·T mismatches and, according to the
nearest-neighbor model, seven of them are linearly independent.
Note that the trimer sequences reported here do not account for
next-nearest-neighbor interactions. To derive the seven linearly
independent trimer sequences one simply adds an arbitrary base pair
to the end of all of the eight dimer nearest-neighbors (in this study
we arbitrarily chose to add a C·G pair to the 3′-end of all of the dimer
sequences). Upon adding the third base pair, two of the trimer
sequences will be the same (GCC/CTG and GTC/CCG), therefore
reducing the number of unique independent trimer sequences to
seven. Duplexes with internal C·T mismatches are expressed as a
linear combination of Watson–Crick dimer nearest-neighbors and
mismatch trimers. Thus, equation 2 can be written as
5-GGACCGACG-3
3-CCTGTCTGC-5 GG GA AC CCG GA AC CG
Init. CC CT TG GTC CT TG GC
5
where the trimer sequence CCG/GTC is accounted for using
CCG
CCC CTC GCC
GTC GTG GCG – CTG
6
Linear regression analysis of C·T mismatch
nearest-neighbors
Thermodynamic parameters derived from averages of the fits and
Tm–1 versus lnCT are equally reliable (16,17,20), thus their
averages were used to determine C·T mismatch contributions to
the total ∆G37, ∆H and ∆S of all 26 duplexes (see equation 4
above). The solution to these 26 equations for seven unknowns
was determined by carrying out multiple linear regression by
singular value decomposition (SVD) analysis (34) using the
program MATHEMATICA v2.1 (Wolfram research) as described
(16,17). The data in the SVD analysis were weighted by their
errors (see below). A similar SVD calculation was performed to
determine ∆H values for the seven trimers. The solutions
obtained were used to calculate ∆S using the equation
∆S = (∆H – ∆G37)/310.15
7
To verify our results, we performed SVD analysis for ∆S and
obtained nearest-neighbor parameters that are in agreement with
those obtained using equation 7.
2696 Nucleic Acids Research, 1998, Vol. 26, No. 11
Error analysis of the data
To determine the error associated with C·T mismatch contributions
to each ∆G37, ∆H and ∆S measurement, we used standard
error propagation methods (35). The uncertainty in measured
∆G37, ∆H and ∆S parameters were assumed to be 4%, 8% and
8% respectively. The measurement errors, in combination with
reported errors for Watson–Crick nearest-neighbors (17), were
propagated to obtain an error estimate for C·T mismatch trimer
contributions using the equation
[ G 37(mismatch)] 2 + [ G 37(measured)] 2 )
ȍ (
G 37
)2
8
NN
where G 37(mismatch) is the propagated error associated with
∆G37(mismatch), G 37(measured) is the uncertainty in the
measured free energy for the duplex (4%) and ȍ (G )2 is the
broadening exponential function and Fourier transformed with a
Silicon Graphics Indigo2Extreme computer with Varian VNMR
software. No baseline correction or solvent subtraction was applied.
3-Trimethylsilyl propionic-2,2,3,3-d4 acid (TSP) was used as the
internal standard for chemical shift reference. One-dimensional
NOE difference spectra were acquired as described above, but
with selective decoupling of individual resonances during the 1 s
recycle delay. Each resonance was decoupled with a power
sufficient to saturate <80% of the signal intensity, so that spillover
artifacts would be minimized. The spectra were acquired in an
interleaved fashion in blocks of 16 scans to minimize subtraction
errors due to long term instrument drift. 3200–6400 scans were
collected for each FID.
NN
37
squared sum of errors for the Watson–Crick nearest-neighbors
represented in the duplex (17). The error in the initiation
parameter is negligible due to large correlation terms (17).
Similar calculations were carried out to obtain H (mismatch) and
S (mismatch). The SVD analysis propagates the mismatch errors to
the determined nearest-neighbor parameters in the variance–
covariance matrix in a rigorous fashion (34).
Resampling analysis of the data
To independently evaluate the error in the obtained C·T mismatch
nearest-neighbors and to point out sequences that are either
outliers in the fit or that have a substantial effect on the solution
obtained by SVD analysis, we performed a resampling analysis
of the data. The solution obtained by performing SVD analysis on
all 26 sequences is over-determined (i.e. 26 equations with seven
unknowns). This resampling analysis has the advantage that it can
determine the uncertainties of C·T mismatch nearest-neighbors
separate of any previous assumption made about the errors in the
measurements (17,36). The resampling analysis was performed
for ∆G37, ∆H and ∆S. We performed 30 resampling trials in
which eight randomly selected sequences were removed. For
each resampling trial, the number of non-zero singular values was
confirmed to be seven. For each nearest-neighbor, the 30 resampling
trials were averaged and standard deviations determined. The
averaged nearest-neighbors from resampling trials were within
round-off error of the values obtained for an SVD analysis with
all 26 sequences. The standard deviations from resampling agree
with the errors propagated in SVD.
1H-NMR
spectroscopy
Oligomers were dissolved in 90% H2O and 10% D2O with 1 M
NaCl, 10 mM disodium phosphate and 0.1 mM Na2EDTA at pH 7.0
or 5.0. Duplex concentrations were between 0.2 and 1.0 mM.
1H-NMR spectra were recorded using a Varian Unity 500 MHz
NMR spectrometer. One-dimensional exchangeable proton NMR
spectra were recorded at 10C using the WATERGATE pulse
sequence with ‘flip-back’ pulse to suppress the water peak (37,38).
Spectra were recorded with the carrier placed at the solvent
frequency and with high power and low power pulse widths of 10.0
and 1800 µs, a sweep width of 12 kHz and a gradient field strength
of 10.0 G/cm and duration of 1 ms. 512–1024 transients were
collected for each spectrum. Data were multiplied by a 4.0 Hz line
RESULTS
Thermodynamics of DNA duplexes with C·T mismatches
Plots of Tm–1 versus lnCT for all the duplexes in this study were
linear (correlation coefficient >0.99; not shown). Thermodynamic
parameters for helix to coil transitions for 28 sequences using
averages of the fits of melting curves and Tm–1 versus lnCT plots
are listed in Table 1. A widely used method for determining
applicability of the two-state model to melting curves is
comparison of the ∆H values obtained from the averages of the
fits and the Tm–1 versus lnCT plots. If the ∆H parameters from
both methods agree within 15%, the duplex to random coil
transition is assumed to be two-state (16,17,20). However, melts
that exhibit agreement of ∆H values of 15% do not necessarily rule
out non-two-state behavior (17,31). Twenty three of the sequences
in Table 1 have a ∆H agreement from the two methods of ≤15%
and showed monophasic transitions, indicating bimolecular twostate behavior. Five duplexes in Table 1 melt with non-two-state
transitions. The non-two-state behavior of these duplexes is
manifested in the >15% disagreement in ∆H values derived by the
two methods. These non-two-state sequences may have the ability
to form alternative conformations, such as hairpins or slipped
duplexes, during the duplex to random coil transition. For duplexes
with two-state transitions, the thermodynamics obtained from the
fits and the Tm–1 versus lnCT plots are equally reliable
(16,17,20,21) and thus their averages are the experimental values
listed in Table 2.
Nearest-neighbor thermodynamics of unique trimer
sequences with internal C·T mismatches
Table 3 lists thermodynamic parameters obtained using SVD
analysis for all 16 unique trimer sequences with internal C·T
mismatches. According to the nearest-neighbor model, seven of
these trimer sequences are linearly independent and can be used
in linear combination to obtain parameters for the other nine
trimer sequences. The errors listed in Table 3 are the standard
deviations from resampling analysis of the data (see Materials and
Methods). These errors are the same as the errors obtained by
propagating the experimental and Watson–Crick nearest-neighbor
errors in the SVD analysis. The parameters listed in Table 3, along
with Watson–Crick nearest-neighbor and initiation parameters
(17), predict the thermodynamics of all 26 duplexes with
two-state thermodynamics (Table 2) with average deviations for
∆G37, ∆H, ∆S and Tm of 0.45 kcal/mol, 5.9 kcal/mol,
18.0 e.u., and 1.9C respectively.
2697
Nucleic Acids
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1998,Vol.
Vol.22,
26,No.
No.111
Nucleic
2697
Table 1. Thermodynamics of duplex formation of oligonucleotides with internal C·T mismatchesa
DNA duplex
–∆G37
(kcal/mol)
1/Tm versus lnCT parameters
–∆H
–∆S
(kcal/mol)
(eu)
Tm
(C)b
–∆G37
(kcal/mol)
Curve fit parameters
–∆H
–∆S
(kcal/mol)
(eu)
Molecules with two-state transitions
CGTCCGTCC
6.73 ± 0.32
56.5 ± 1.3
160.4 ± 3.1
42.9
6.68 ± 0.11
60.1 ± 1.6
172.3 ± 5.4
CGTGCCTCC
6.75 ± 0.15
57.1 ± 0.6
162.2 ± 1.5
43.0
6.75 ± 0.04
59.1 ± 1.0
168.6 ± 3.2
(6.28 ± 0.22)
(53.6 ± 0.9)
(152.5 ± 2.1)
(40.7)
(6.31 ± 0.03)
(53.3 ± 1.4)
(151.4 ± 4.7)
GGACCCTCG
6.23 ± 0.21
53.2 ± 0.9
151.4 ± 2.1
40.3
6.21 ± 0.03
55.0 ± 1.1
157.3 ± 3.4
GGACCGACG
6.60 ± 0.51
54.5 ± 2.0
154.3 ± 4.8
42.4
6.56 ± 0.10
59.1 ± 1.0
169.4 ± 3.1
GGAGCCACG
6.59 ± 0.24
57.0 ± 1.0
162.5 ± 2.5
42.0
6.58 ± 0.04
59.0 ± 1.6
169.0 ± 5.1
CACAGCAGGTC
7.74 ± 0.22
65.6 ± 1.0
186.6 ± 2.4
47.0
7.75 ± 0.04
62.6 ± 3.9
176.8 ± 12.4
CATGACGCTAC
8.44 ± 0.86
73.8 ± 4.0
210.7 ± 10.1
49.1
8.61 ± 0.18
85.9 ± 4.3
249.3 ± 13.5
(7.93 ± 0.33)
(67.5 ± 1.5)
(192.2 ± 3.7)
(47.5)
(8.10 ± 0.23)
(80.7 ± 3.6)
(234.0 ± 10.9)
8.00 ± 0.46
71.9 ± 2.1
206.1 ± 5.4
47.3
8.01 ± 0.07
73.1 ± 1.9
209.8 ± 6.0
CATGATGCTAC
CATGTCACTAC
6.98 ± 0.10
66.0 ± 0.5
190.3 ± 1.3
43.3
6.99 ± 0.03
66.6 ± 2.6
192.2 ± 8.3
CATGTTACTAC
6.91 ± 0.15
65.5 ± 0.7
188.8 ± 1.7
43.0
6.92 ± 0.02
65.6 ± 1.8
189.1 ± 6.0
GAACGCTGTCC
8.29 ± 0.41
62.6 ± 1.6
175.1 ± 4.0
50.5
8.45 ± 0.13
64.8 ± 4.6
181.7 ± 14.4
GACCTCCTGTG
7.56 ± 0.23
66.5 ± 1.0
190.0 ± 2.6
46.1
7.55 ± 0.05
61.7 ± 2.5
174.6 ± 8.2
GATCATTGTAC
7.04 ± 0.44
67.2 ± 2.0
194.0 ± 5.0
43.4
7.01 ± 0.09
72.9 ± 3.2
212.5 ± 10.1
GATGTCTGTAC
6.61 ± 0.29
65.5 ± 1.4
189.8 ± 3.5
41.5
6.58 ± 0.04
69.8 ± 2.4
203.8 ± 7.6
GCTAGCAATCC
7.27 ± 0.17
67.3 ± 0.8
193.4 ± 2.0
44.8
7.25 ± 0.10
59.8 ± 2.2
169.6 ± 7.0
CGCCAGAGCCGG
6.73 ± 0.61
43.7 ± 1.9
119.0 ± 4.2
44.7
6.68 ± 0.13
49.9 ± 3.0
139.3 ± 9.4
CGCTAGAGTCGG
6.44 ± 0.29
46.7 ± 1.0
129.8 ± 2.3
42.2
6.43 ± 0.05
48.6 ± 2.0
136.1 ± 6.5
GGCCGAGACCGC
7.56 ± 0.59
65.2 ± 2.6
186.0 ± 6.5
46.2
7.56 ± 0.06
63.1 ± 3.1
179.2 ± 9.9
GGCTGAGATCGC
7.34 ± 0.47
60.9 ± 2.0
172.6 ± 4.8
45.7
7.34 ± 0.06
56.3 ± 1.4
157.9 ± 4.6
CGACCATATGTTCG
6.29 ± 0.32
53.1 ± 1.3
151.0 ± 3.2
40.6
6.37 ± 0.07
51.1 ± 5.6
144.1 ± 10.1
CGTCTCATGATACG
7.28 ± 0.29
79.1 ± 1.6
231.5 ± 4.4
43.4
7.23 ± 0.09
69.5 ± 3.3
200.6 ± 10.9
(6.59 ± 0.37)
(73.5 ± 2.0)
(215.7 ± 5.4)
(41.0)
(6.66 ± 0.17)
(61.2 ± 4.6)
(176.0 ± 15.3)
CTCCACATGTTGAG
6.80 ± 0.34
72.1 ± 3.5
210.5 ± 9.2
41.9
6.78 ± 0.15
61.9 ± 4.1
177.8 ± 13.5
(6.39 ± 0.51)
(59.8 ± 2.3)
(172.2 ± 5.8)
(40.8)
(6.48 ± 0.11)
(54.6 ± 5.8)
(155.4 ± 18.9)
6.49 ± 0.33
71.1 ± 1.8
208.3 ± 4.7
40.6
6.52 ± 0.12
60.4 ± 4.1
173.6 ± 13.7
CTCTCATATGCGAG
Molecules with non-two-state transitions
CGAGCGTCC
6.20 ± 0.63
65.8 ± 3.1
192.1 ± 8.1
39.5
6.38 ± 0.17
51.0 ± 2.0
143.8 ± 6.2
GAACGCAGTCC
6.59 ± 0.96
26.3 ± 1.8
63.6 ± 2.9
48.2
6.51 ± 0.30
38.3 ± 7.9
102.6 ± 24.8
GATCTTTGTAC
7.14 ± 0.26
67.3 ± 1.2
194.1 ± 3.1
43.9
7.11 ± 0.15
81.0 ± 3.4
238.3 ± 10.6
CTCTATGGTACTGC
7.50 ± 0.59
88.8 ± 3.6
262.3 ± 9.6
43.5
7.45 ± 0.15
68.3 ± 1.4
196.3 ± 4.8
GCATCTGCGGCTAG
10.28 ± 2.10
46.7 ± 5.6
117.4 ± 11.3
71.0
9.78 ± 0.44
39.1 ± 5.1
94.4 ± 15.1
aListed in alphabetical order and by oligomer length. For each DNA duplex only the top strand is shown. Underlined residues indicate the position of a C·T mismatch.
Molecules listed as two-state had ∆H agreement within 15% by two different methods. Molecules listed as non-two-state had ∆H disagreement of >15%. Solutions
are 1 M NaCl, 20 mM sodium cacodylate, 0.5 mM Na2EDTA, pH 7.0. Values reported in parentheses were obtained under the same solution conditions as above
except at pH 5.0. Errors are standard deviations from the regression analysis of the melting data. Extra significance figures are given to allow accurate calculation
of ∆G37 and Tm.
bCalculated for 10–4 M oligomer concentration for self-complementary sequences and 4 × 10–4 M for non-self-complementary sequences.
2698 Nucleic Acids Research, 1998, Vol. 26, No. 11
Table 2. Experimental and predicted thermodynamics of oligonucleotides with C·T mismatchesa
DNA duplex
Ref.b
–∆G37 (kcal/mol)c
Expt.
Predicted
–∆H (kcal/mol)c
Expt.
Predicted
–∆S (e.u)c
Expt.
Predicted
Tm (C)d
Expt.
Predicted
Molecules with two-state transitions
CAAACAAAG
(24)
3.27
3.38
53.2
46.0
161.0
137.2
23.6
22.7
CAAATAAAG
(24)
3.17
3.07
50.0
47.7
151.0
143.6
22.2
21.5
6.70
6.51
58.3
53.9
166.4
152.5
42.6
42.4
CGTCCGTCC
CGTGCCTCC
6.75
5.92
58.1
43.8
165.4
122.1
42.9
38.8
GGACCCTCG
6.22
5.77
54.1
46.6
154.4
131.5
40.2
37.9
GGACCGACG
6.58
6.51
56.8
53.9
161.8
152.5
42.0
42.4
GGAGCCACG
6.58
5.92
58.0
43.8
165.7
122.1
41.9
38.8
CACAGCAGGTC
7.74
8.15
64.1
62.1
181.7
173.8
47.3
50.1
CATGACGCTAC
8.52
7.62
79.9
66.2
230.0
188.6
48.5
46.8
CATGATGCTAC
8.01
7.31
72.5
67.4
207.9
193.4
47.3
45.2
CATGTCACTAC
6.99
6.28
66.3
62.0
191.2
179.5
43.3
40.3
CATGTTACTAC
6.92
6.28
65.5
62.0
189.0
179.5
43.0
40.3
GAACGCTGTCC
8.37
8.63
63.7
66.9
178.4
187.6
50.7
51.8
GACCTCCTGTG
7.56
7.57
64.1
59.0
182.3
165.7
46.4
47.5
GATCATTGTAC
7.03
6.51
70.1
64.9
203.2
187.9
43.1
41.6
GATCTCTGTAC
6.59
6.01
67.6
63.7
196.8
185.7
41.3
39.1
GCTAGCAATCC
7.26
7.07
63.5
60.4
181.5
171.9
44.9
44.4
CGCCAGAGCCGG
6.71
7.35
46.8
54.9
129.1
153.4
44.0
46.6
CGCTAGAGTCGG
6.44
7.35
47.7
55.4
132.9
155.0
42.0
46.5
GGCCGAGACCGC
7.56
7.77
64.2
58.6
182.6
163.8
46.4
48.6
GGCTGAGATCGC
7.34
8.04
58.6
63.4
165.3
178.3
46.1
49.3
CGACCATATGTTCG
6.33
6.58
52.1
64.2
147.5
185.9
40.9
41.2
CGTCTCATGATACG
7.25
7.84
74.3
78.4
216.0
227.4
43.7
45.9
CTCCACATGTTGAG
6.78
6.72
67.0
72.4
194.2
211.6
42.2
41.8
CTCTCATATGCGAG
6.51
6.00
65.7
68.8
190.9
202.3
41.0
38.7
9.70
10.13
98.4
99.4
286.0
287.4
50.2
52.0
CAACTTGATATTAATA
(25)
Molecules with non-two-state transitions
CGAGCGTCC
6.29
6.35
58.4
50.2
168.0
141.2
40.3
41.6
GAACGCAGTCC
7.12
6.32
74.2
62.0
216.2
179.3
43.2
40.6
GATCTTTGTAC
6.56
8.44
32.3
64.0
83.1
179.0
45.7
51.2
CTCTATGGTACTGC
7.48
7.76
78.6
74.2
229.3
214.0
44.3
46.3
GCATCTGCGGCTAG
10.03
9.87
42.9
75.6
105.9
211.8
72.1
55.4
aListed in alphabetical order and by oligomer length. For each DNA duplex only the top strand is shown. Underlined residues indicate the position
of a C·T mismatch. Experimental values are the averages of the Tm–1 versus lnCT and the curve fit parameter given in Table 1.
without a literature reference are from Table 1 of this work.
cStandard errors for experimental ∆G , ∆H and ∆S are assumed to be 4, 8 and 8% respectively.
37
dCalculated for 10–4 M oligomer concentration for self-complementary sequences and 4 × 10–4 M for non-self-complementary sequences.
bSequences
Non-unique C·T mismatch nearest-neighbor thermodynamics
As stated previously, analysis of internal C·T mismatches in terms
of dimer sequences results in eight nearest-neighbors that are not
a unique solution. The non-uniqueness of these dimer sequences
results from having all C·T mismatches located internally
(17,33). Table 4 lists nearest-neighbor parameters for dimer
sequences with C·T mismatches obtained by fitting the data to
eight parameters. The eight dimer parameters listed in Table 4 are
an alternative representation of the seven trimer parameters listed
in Table 3. However, in the SVD analysis of eight dimer
sequences, the number of non-zero singular values is seven,
indicating that the stacking matrix is rank deficient and that the
parameters are non-unique. To clarify the non-uniqueness of the
parameters in Table 4 one could show that a linear combination
of the parameters in Table 4 can be used to derive parameters for
2699
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the seven linearly independent trimer sequences in Table 3, but
not vice versa unless an eighth parameter is given (SVD assumes
the eighth parameter is zero) (14). Nonetheless, the parameters in
Tables 3 and 4 result in the same predictions and, thus, one could
use either representation of the data, keeping in mind that both
apply only to internal C·T mismatches.
Table 3. Nearest-neighbor thermodynamic parameters for
16 trimer sequences with internal C·T mismatches in 1 M
NaCla
Propagation
sequence
∆H
(kcal/mol)
∆S
(e.u)
∆G37
(kcal/mol)
ACC/TTG
5.9 ± 1.3
13.8 ± 2.6
1.62 ± 0.10
CCC/GTG
4.4 ± 1.2
9.0 ± 2.5
1.60 ± 0.13
GCA/CTT
3.3 ± 1.1
6.2 ± 2.2
1.37 ± 0.12
GCC/CTG
7.5 ± 1.5
19.0 ± 3.0
1.60 ± 0.13
1.02 ± 0.11
Seven linearly independent trimers
GCG/CTC
0.8 ± 1.2
–0.7 ± 2.5
GCT/CTA
1.1 ± 1.3
–0.8 ± 2.0
1.35 ± 0.12
TCC/ATG
6.4 ± 1.3
14.3 ± 2.9
1.95 ± 0.14
The nine other trimer contextsb
ACA/TTT
1.7 ± 1.4
1.0 ± 2.0
ACG/TTC
–0.8 ± 1.7
–5.9 ± 3.0
1.39 ± 0.11
1.04 ± 0.14
ACT/TTA
–0.5 ± 1.3
–6.0 ± 2.8
1.37 ± 0.15
CCA/GTT
0.2 ± 1.1
–3.8 ± 2.2
1.37 ± 0.10
CCG/GTC
–2.3 ± 1.4
–10.7 ± 2.2
1.02 ± 0.13
CCT/GTA
–2.0 ± 1.5
–10.8 ± 2.3
1.35 ± 0.14
TCA/ATT
2.2 ± 1.3
1.5 ± 2.1
1.72 ± 0.13
TCG/ATC
–0.3 ± 1.3
–5.4 ± 3.0
1.37 ± 0.12
TCT/ATA
0.0 ± 1.5
–5.5 ± 2.9
1.70 ± 0.15
aErrors
are resampling standard deviations (see text).
bThese other contexts can be derived from linear combinations of
the seven linearly independent trimer sequences (see text).
These parameters are not applicable to terminal or penultimate
C•T mismatches.
Table 4. Nearest-neighbor thermodynamics of C·T
mismatches in 1 M NaCla
Dimer
sequence
∆H
(kcal/mol)
∆S
(e.u)
∆G37
(kcal/mol)
AC/TT
0.7
0.2
0.64
AT/TC
–1.2
–6.2
0.73
CC/GT
–0.8
–4.5
0.62
CT/GC
–1.5
–6.1
0.40
GC/CT
2.3
5.4
0.62
GT/CC
5.2
13.5
0.98
TC/AT
1.2
0.7
0.97
TT/AC
1.0
0.7
0.75
2699
Thermodynamics of C·T mismatches at pH 5.0
To test the thermodynamic effects of protonation of a C·T
mismatch (i.e. C+·T versus C·T) thermodynamic measurements
were made on four duplexes with C·T mismatches at pH 5.0 and
pH 7.0. The pKa of protonation for cytosine in the context of a C·T
mismatch has been reported to be ∼5.7 (39), thus, at pH 5.0, ∼66%
of C·T mismatches should be protonated. Four sequences were
selected to represent different C·T mismatch nearest-neighbor
contexts. On average, for the four C·T mismatch-containing
sequences tested for pH effects, changing the pH from 7.0 to 5.0
decreased the stability of the duplex by 0.3 kcal/mol for ∆G37
and 1.1C for the Tm. The data obtained for these four sequences
suggest that the thermodynamics of C·T mismatches at pH 5.0 are
slightly less stable than at pH 7.0.
NMR and pairing geometry of C·T and C+·T mismatches
C·T mismatches have been proposed to form at least four different
structures depending on sequence context and solution conditions
(Fig. 1; 39–42). To determine the pairing geometry for C·T
mismatches in this study, one-dimensional exchangeable proton
NMR spectra of five DNA duplexes with different C·T mismatch
contexts were acquired at pH 7.0 and 5.0. Figures 2 and 3 show
a representative imino region (9–15 ppm) of two of the duplexes
studied containing C·T mismatches at pH 7.0 and 5.0. Resonances
between 12–13 and 13–15 ppm are usually the imino protons of
canonical Watson–Crick G·C and A·T pairs. At pH 7.0, an imino
peak is observed around 11.5 ppm (Figs 2a and 3a) which
broadens out at pH 5.0 (Figs 2b and 3b). Irradiation of this
resonance did not show any observable NOEs (not shown),
probably due to rapid chemical exchange with water. Previous
structural studies on C·T and C·U mismatches in DNA and RNA
showed that at neutral pH these mismatches can pair with two
hydrogen bonds, one of which, due to the repulsion of the
carbonyl groups of the cytosine and thymine (43), is possibly
mediated via a water molecule (Fig. 1b; 39–42). Our data are most
consistent with NMR observations on C·T mismatches at neutral pH
and, thus, we tentatively assign the resonance at 11.5 ppm as the
imino proton of thymine hydrogen bonded to N3 of cytosine via
a water molecule (39,40,42). At pH 5.0, the protonation of N3 of
cytosine results in a change in the pairing geometry of the C·T
mismatch which broadens the imino resonance of the thymine in
the C·T mismatch (11.5 ppm). This broadening of the imino
resonance might be a result of chemical exchange between
protonated and non-protonated C·T mispairs. Previous structural
studies of C·T mismatches under acidic conditions suggest that the
imino proton of thymine becomes hydrogen bonded to the carbonyl
group of cytosine, possibly via a water molecule, making it
exchange faster with water (Fig. 1c and d; 39,40). In contrast, C·C
and A·C in RNA (44) and in DNA (H.T.Allawi and J.SantaLucia Jr,
unpublished results) are often stabilized at acidic pH.
DISCUSSION
aThese parameters are a linear least squares fit of the data for
Applicability of the nearest-neighbor model to internal C·T
mismatches
a singular matrix with a rank of 7. These parameters make
predictions that are the same as those made by the parameters
listed in Table 3. Linear combinations of the parameters in this
table give the parameters in Table 3.
These parameters are not applicable to terminal or penultimate
C•T mismatches.
Table 2 compares experimental results of 26 duplexes with C·T
mismatches with predictions made by the parameters listed in
Table 3 (or Table 4) and Watson–Crick nearest-neighbor parameters
(17). For single mismatches in DNA, we have previously shown
that a nearest-neighbor model can accurately predict duplexes
2700 Nucleic Acids Research, 1998, Vol. 26, No. 11
Figure 1. Four hydrogen bonded structures of the C·T mispair at neutral pH
(a and b) and at acidic pH (c and d).
with internal G·A and G·T with average deviations for ∆G37,
∆H, ∆S and Tm of 5.0%, 8.0%, 8.0%, and 1.5C respectively
(17,21). In this study, we find that analysis of C·T mismatch
contributions to duplex stability in terms of a nearest-neighbor
model results in parameters that predict the thermodynamics of
sequences with two-state transitions with an average deviation for
∆G37, ∆H, ∆S and Tm of 6.4%, 9.9%, 10.6% and 1.9C
respectively. These average deviations are slightly higher than
what was observed for G·A and G·T mismatches (17,21).
Nonetheless, considering how unstable C·T mismatches are, one
might expect that C·T mismatches are capable of disrupting
double-helical DNA in a fashion that may extend to next-nearestneighboring Watson–Crick pairs. However, results from this
study suggest that if there are any next-nearest-neighbor effects
for C·T mismatches they are very small and can be neglected.
Hence, the nearest-neighbor parameters in Tables 3 and 4 make
predictions that are adequate for most applications. An alternative
way to test the applicability of the nearest-neighbor model is to
synthesize oligonucleotides with different sequences but the same
nearest-neighbor composition (17,45–47). In this study, three
pairs of duplexes have the same nearest-neighbor composition
(Tables 1 and 2). For example, the duplexes CGTGCCTCCGGAGTCACG and GGAGCCACGCGTGTCTCC have
different sequences but the same nearest-neighbors and their ∆G37,
∆H, ∆S and Tm agree within 0.17 kcal/mol, 0.1 kcal/mol, 0.3 e.u.,
and 1.0C respectively. The average deviation from the mean
between the three pairs of duplexes with the same nearest-neighbors
for ∆G37, ∆H, ∆S and Tm are 0.06 kcal/mol, 0.4 kcal/mol,
1.2 e.u., and 0.3C respectively.
Trends in C·T mismatch thermodynamics
Trimer mismatch free energy (∆G37) contributions for internal
C·T mismatches vary weakly, depending on the mismatch
orientation and context (Tables 3 and 4). The most stable trimer
sequences (GCG/CTC and CCG/GTC) destabilize the duplex by
+1.02 kcal/mol and the least stable trimer (TCC/ATG) destabilizes
the duplex by +1.95 kcal/mol. This range of 0.93 kcal/mol for
∆G37 indicates that there is a weak stacking contribution to
Figure 2. 500 MHz 1H-NMR spectra of the exchangeable imino region
(9–15 ppm) in 1 M NaCl, 10 mM disodium phosphate and 0.1 mM Na2EDTA
at 10C in 90% H2O/10% D2O of CATGTTACTAC•GTACTCACATG at
(a) pH 7.0 and (b) pH 5.0.
Figure 3. 500 MHz 1H-NMR spectra of the exchangeable imino region
(9–15 ppm) in 1 M NaCl, 10 mM disodium phosphate and 0.1 mM Na2EDTA
at 10C in 90% H2O/10% D2O of (CGTCTCATGATACG)2 at (a) pH 7.0 and
(b) pH 5.0.
stability of a C·T mismatch. For trimer sequences with the
cytosine of the C·T mismatch on the top strand, the general trend
for the 5′-end closing Watson–Crick pair (with decreasing order
of stability) is G·C ≈ C·G > A·T >> T·A. However, when the
thymine of the C·T mismatch is on the top strand (i.e. T·C), the
trend on the 5′-end becomes (with decreasing order of stability)
C·G > A·T ≈ T·A > G·C. Close inspection of these trends reveals
an interesting result. Generally, a G·C base pair (which has three
hydrogen bonds) is expected to have a stabilizing effect on
duplexes that is larger than an A·T pair (which has two hydrogen
bonds). However, G·C pairs stacked on the 5′-end of a T·C
mismatch destabilize the duplex by 0.98 kcal/mol and A·T pairs
2701
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Nucleic
stacked on the 5′-end of a T·C mismatch destabilize the duplex by
0.73 kcal/mol. Therefore, in this case, a 5′ A·T pair stabilizes T·C
mismatches more than does a 5′ G·C. Thus, stacking interactions,
more than hydrogen bonding, play a major role in the stability of
duplexes with internal C·T mismatches. This is also evident when
a G·C pair stacked on a T·C mismatch (GT/CC) is compared with
a C·G (CT/GC), which are destabilizing by 0.98 and 0.40 kcal/mol
respectively (Table 4).
Comparison of thermodynamics of C·T mismatches and
Watson–Crick pairs
No correlation is observed when comparing thermodynamics of
trimer sequences with internal C·T mismatches with the
corresponding trimer sequences with either G·C or A·T Watson–
Crick base pairs (17). Free energies of Watson–Crick trimer
sequences with a central A·T or G·C pair vary over a range of
2.95 kcal/mol, whereas the range of trimer sequences with
internal C·T mismatches vary over 0.93 kcal/mol in ∆G37. The
most stable Watson–Crick trimer sequence is GCG/CGC (∆G37
= –4.41 kcal/mol) and the least stable is ATA/TAT (∆G37 =
–1.46 kcal/mol) (17). For internal C·T mismatches, the most
stable C·T trimer sequence contexts are GCG/CTC and
CCG/GTC (+1.02 kcal/mol) and is the same context as the most
stable Watson–Crick sequence (GCG/CGC). However, the trimer
sequence TCC/ATG, which is the least stable C·T context, is
different than the least stable Watson–Crick sequence (ATA/TAT).
Comparison of C·T, G·T and G·A mismatch thermodynamics
Comparison of internal C·T mismatches thermodynamics (Table 3)
with previously published parameters for internal G·A (21) and
G·T (17) mismatch thermodynamics indicates that C·T mismatches
are among the most unstable mismatches in DNA consistent with
previous observations (23,24). The most stable C·T trimer
sequences are GCG/CTC and CCG/GTC (∆G37 of +1.02 kcal/
mol) and the most stable G·A or G·T trimer sequences are
GGC/CAG and CGC/GTG (∆G37 –0.78 and –1.05 kcal/mol
respectively). Moreover, the least stable C·T trimer sequence is
TCC/ATG (∆G37 +1.95 kcal/mol) and the least stable G·A or
G·T trimer sequences are TGA/AAT and AGA/TTT (∆G37
+1.16 and +1.05 kcal/mol respectively). The average free energy
contribution of all 16 unique trimer sequences with internal C·T
mismatches is +1.43 kcal/mol. Average internal G·A and G·T
mismatch free energy contributions for all 16 unique trimer
sequences, on the other hand, are +0.17 and +0.05 kcal/mol
respectively. Furthermore, stabilities of G·A and G·T mismatches
are spread over a range of 1.94 and 2.10 kcal/mol respectively,
while C·T mismatch stabilities are spread over a range of
0.93 kcal/mol indicating that, while contributions of internal C·T
mismatch thermodynamics depend slightly on the neighboring
bases, their thermodynamics are not as sensitive to the surrounding
base pair context as in G·A and G·T mismatches.
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